Abstract
The viral RNA-dependent RNA polymerase (RdRp; L protein) of Rift Valley fever virus (RVFV; family Bunyaviridae) is a 238 kDa protein that is crucial for the life cycle of the virus, as it catalyses both transcription of viral mRNAs and replication of the tripartite genome. Despite its importance, little is known about the intracellular distribution of the polymerase or its other roles during infection, primarily because of lack of specific antibodies that recognize L protein. To begin to address these questions we investigated whether the RVFV (MP12 strain) polymerase could tolerate insertion of the V5 epitope, as has been previously demonstrated for the Bunyamwera virus L protein. Insertion of the 14 aa epitope into the polymerase sequence at aa 1852 resulted in a polymerase that retained functionality in a minigenome assay, and we were able to rescue recombinant viruses that expressed the modified L protein by reverse genetics. The L protein could be detected in infected cells by Western blotting with anti-V5 antibodies. Examination of recombinant virus-infected cells by immunofluorescence revealed a punctate perinuclear or cytoplasmic distribution of the polymerase that co-localized with the nucleocapsid protein. The generation of RVFV expressing a tagged RdRp will allow detailed examination of the role of the viral polymerase in the virus life cycle.
Highlights
Rift Valley fever virus (RVFV) is a mosquito-borne pathogen of both livestock and humans that is found primarily in sub-Saharan Africa and the Arabian Peninsula
The RNA-dependent RNA polymerase (RdRp) of negative-sense RNA viruses plays a crucial role in the virus life cycle, including replication of the viral genome and transcription of viral mRNAs (Ortın & Parra, 2006)
As with other bunyaviruses, detailed study of the RVFV L protein has been impeded by the lack of antibodies that can detect the authentic L protein in infected cells by techniques such as Western blotting or immunofluorescence
Summary
Rift Valley fever virus (RVFV) is a mosquito-borne pathogen of both livestock and humans that is found primarily in sub-Saharan Africa and the Arabian Peninsula. 1.7 kb) encodes the nucleocapsid (N) protein and a nonstructural protein (NSs) in an ambisense manner. The N protein is translated from a subgenomic mRNA that is transcribed from the genomic RNA, while NSs is translated from a subgenomic mRNA that is transcribed from the antigenomic (replicative-intermediate) RNA. 6.4 kb) encodes the viral RNA-dependent RNA polymerase (RdRp or L protein). The RNA segments are encapsidated by N protein to form ribonucleoprotein (RNP) complexes, the functional templates for viral RNA synthesis (Ortın & Parra, 2006)
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